Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), is deeply entrenched in the human population causing ~8 million new active TB cases and ~1.5 million deaths per year. Treating TB with currently available antibiotics remains problematic, where current regimens require a multidrug, 6-9 month course. This long course leads to non-compliance and is fueling the emergence of multidrug resistant Mtb. Stemming the ongoing TB epidemic requires a variety of new approaches, including the development of new drugs that: i) shorten the course of TB treatment, and ii) are effective against antimicrobial resistant Mtb. Mtb alters its physiology in response to host immune pressures thus enabling the bacterium to remain viable in humans for decades. Mtb is an intracellular pathogen that resides within macrophages (M?), a host immune cell that kills most other bacteria. Following infection, the M? releases immune modulators that orchestrate the formation of a granuloma around the infected M?. The granuloma limits the availability of nutrients and oxygen to the bacterium and drives Mtb to realign its gene expression and physiology to support a non-replicative persistent (NRP) state. During infection Mtb is exposed to the antimicrobial reactive nitrogen intermediate (RNI) nitric oxide (NO). One mechanism used by Mtb to counter NO stress is the Mtb proteasome. Mtb mutants lacking a proteasome are highly sensitive to NO and have reduced virulence in mice. In vitro sensitivity to NO is enhanced during NRP and the proteasome is required during persistent infection in mice, supporting an important role of the proteasome in maintaining a chronic infection. A major obstacle in the development of effective Mtb proteasome inhibitors is their inherent host toxicity. This host toxicity is due to the mechanism of proteasome inhibition, which involves the covalent binding of the electrophilic suicide substrate to the catalytic site of the proteasome. This type of binding i directly correlated to their lack of specificity, low systemic tissue distribution and severe off-target effects. We have developed a class of proteasome inhibitors that regulate the proteasome via a mechanistically distinct, non- covalent, non-competitive, protein:ligand interaction, which effectively inhibits Mtb growth with no apparent host toxicity. Importantly, we found that inhibition of Mtb growth is amplified under NO stress. These agents are excellent candidates to pursue as Mtb antimicrobials. In the R21 phase of this proposal we will define the mechanism of binding, optimize the scaffolds potency for the Mtb proteasome (Aim 1) and characterize the bioactivity in Mtb (Aim 2). The successful completion of these aims will support continued development of a first-in-its-class agent to target chronic TB, and evaluate drug-resistant TB, in vivo pharmacological requirements and optimize the in vivo efficacy of prioritized compounds in order to obtain in vivo proof-of-principle of this novel approach to combat TB.